Constitutively Active Mutant of the Mitogen-activated Protein Kinase Kinase MEK1 Induces Epithelial Dedifferentiation and Growth Inhibition in Madin-Darby Canine Kidney-C7 Cells

doi:10.1074/jbc.272.17.11426

Constitutively Active Mutant of the Mitogen-activated Protein Kinase Kinase MEK1 Induces Epithelial Dedifferentiation and Growth Inhibition in Madin-Darby Canine Kidney-C7 Cells*

From the Department of Physiology, University of Innsbruck, A-6010 Innsbruck, Austria and the
^§ Department of Pharmacology, University College Dublin, Dublin 4, Ireland

^‡ To whom correspondence should be addressed. Tel.: 512-507-3777; Fax: 512-507-2853; E-mail: herbert.schramek{at}uibk.ac.at

Abstract

Overexpression of a constitutively active mitogen-activated protein kinase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the present study, we used a constitutively active MAPK/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the function of the highly conserved MEK1-ERK2 signaling module in renal epithelial cell differentiation and proliferation. Stable expression of constitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serum-stimulated ERK1 and ERK2 phosphorylation as well as ERK2 activation when compared with mock-transfected cells. In both mock-transfected and CA-MEK1-transfected MDCK-C7 cells, basal and serum-stimulated ERK1 and ERK2 phosphorylation was almost abolished by the synthetic MEK inhibitor PD098059. Increased ERK2 activation due to stable expression of CA-MEK1 in MDCK-C7 cells was associated with epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increased vimentin expression. In addition, we obtained a delayed and reduced serum-stimulated cell proliferation in CA-MEK1-transfected cells (4.6-fold increase in cell number/cm² after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm² after 5 days). This result was confirmed by flow cytometric DNA analysis showing that stable expression of CA-MEK1 decreased the proportion of MDCK-C7 cells moving from G₀/G₁ to G₂/M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serum-stimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells. Thus, the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation thereby leading to an attenuation of MDCK-C7 cell proliferation.

Footnotes

↵* This work was supported by the Austrian Science Foundation, Grant P11125-MED (to H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ERK

extracellular signal-regulated kinase

MAPK

mitogen-activated protein kinase

JNK

c-Jun NH₂-terminal kinase

MEK

MAPK/ERK kinase

MEKK

MEK kinase

MBP

myelin basic protein

MDCK

Madin-Darby canine kidney

PC12

adrenal pheochromocytoma 12

FCS

fetal calf serum

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

BSA

bovine serum albumin

CA

constitutively active

FACS

fluorescence-activated cell sorter

MEM

minimal essential medium

HA

hemagglutinin.
- Received December 20, 1996.
- Revision received February 18, 1997.